U.S. patent number 5,472,842 [Application Number 08/132,808] was granted by the patent office on 1995-12-05 for detection of amplified or deleted chromosomal regions.
This patent grant is currently assigned to The Regents of the University of California. Invention is credited to Joe W. Gray, Daniel Pinkel, Trond Stokke.
United States Patent |
5,472,842 |
Stokke , et al. |
December 5, 1995 |
**Please see images for:
( Certificate of Correction ) ** |
Detection of amplified or deleted chromosomal regions
Abstract
The present invention relates to in situ hybridization methods
for the identification of new chromosomal abnormalities associated
with various diseases. In particular, it provides probes which are
specific to a region of amplification in chromosome 20.
Inventors: |
Stokke; Trond (San Francisco,
CA), Pinkel; Daniel (Walnut Creek, CA), Gray; Joe W.
(San Francisco, CA) |
Assignee: |
The Regents of the University of
California (Oakland, CA)
|
Family
ID: |
22455696 |
Appl.
No.: |
08/132,808 |
Filed: |
October 6, 1993 |
Current U.S.
Class: |
435/6.14;
536/24.31 |
Current CPC
Class: |
C12Q
1/6827 (20130101); C12Q 1/6841 (20130101); Y10S
435/81 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C12Q 001/68 (); C12N 015/11 () |
Field of
Search: |
;435/6 ;536/24.31 |
Foreign Patent Documents
Other References
Matthews et al, Anal. Biochem., v. 169, Feb. 1988, 1-25. .
Cremer et al, Hum. Genet., v. 80, 1988, 235-246. .
Lichter et al, Hum. Genet., v. 80, 1988, 224-234. .
Kallioniemi et al, Proc. Natl. Acad. Sci., v. 91, 1994, pp.
2156-2160. .
Kallioniemi et al, Science, v. 258, Oct. 30, 1992, 818-821. .
Wiegart et al, Nucleic Acids, Res, v. 19, 1991, 3237-3241. .
J. N. Lucas et al. "Translocations between two specific human
chromosomes detected by three-color `chromosome painting`"
Cytogenet Cell Genet vol. 62 pp. 11-12 (1993). .
Annika Lindblom et al. "Deletions on Chromosome 16 in Primary
Familial Breast Carcinomas Are Associated with Development of
Distant Metastases" Cancer Research vol. 53, pp. 3707-3711, Aug.
15, 1993. .
Ulf S. R. Bergerheim et al. "Deletion Mapping of Chromosomes 8, 10,
and 16 in Human Prostatic Carcinoma" Genes, Chromosomes &
Cancer vol. 3, pp. 215-220 (1991). .
Elisabeth Blennow et al. "Complete characterization of a large
marker chromosome by reverse and forward chromosome painting" m
Genet (1992) vol. 90, pp. 371-374. Received: 15 Jun. 1992/Revised:
6 Jul. 1992. .
Stefan Joos et al. "Detection of amplified DNA sequences by reverse
chromosome painting using genomic tumor DNA as probe" m Genet
(1993) vol. 90, pp. 584-589. Received: 30 Oct. 1992. .
Anne Kallioniemi et al. "Comparative Genomic Hybridization for
Molecular Cytogenetic Analysis of Solid Tumors" Science vol. 258,
Oct. 30, 1992 pp. 818-821. .
Peter Lichter et al. "High-Resolution Mapping of Human Chromosome
11 by in Situ Hybridization with Cosmid Clones" Science vol. 247
Jan. 5, 1990 pp. 64-69. .
Anne Kallioniemi et al. "ERBB2 amplification in breast cancer
analyzed by fluorescence in situ hybridization" Proc. Acad. Nat.
Sci. 89:5321-5325 (1992). .
Pinkel et al. "Fluorescence in situ hybridization with human
chromosome-specific libraries: Detection of trisomy 21 and
translocations of chromosome 4" Proc. Acad. Nat. Sci. 85:9138-9142
(1988). .
Stokke, et al. "Genetic characterization of 20q amplication in
human breast cancer" Abstract submitted to the 43rd Annual Meeting
of the American Society of Human Genetics (Appendix 2)..
|
Primary Examiner: Fleisher; Mindy B.
Assistant Examiner: Ketter; James
Attorney, Agent or Firm: Townsend and Townsend and Crew
Government Interests
This invention was made with Government support under Contract No.
W-7405-ENG-48 awarded by the Department of Energy and Grant No.
CA-45919 awarded by the National Institutes of Health. The
Government has certain rights in this invention.
Claims
What is claimed is:
1. A method of detecting a chromosome abnormality in a preselected
chromosome, the method comprising:
providing a mapped library of labeled probes specific to the
chromosome;
contacting a chromosome sample from a patient with the library
under conditions in which the probes bind selectively with target
polynucleotide sequences in the sample to form hybridization
complexes;
detecting the hybridization complexes; and
determining the copy number of each complex, thereby detecting the
presence or absence of a chromsome abnormality in the
chromosome.
2. The method of claim 1, wherein the chromosome abnormality is a
deletion.
3. The method of claim 1, wherein the chromosome abnormality is an
amplification.
4. The method of claim 1, wherein the probes are labeled with
digoxigenin or biotin.
5. The method of claim 1, wherein the step of detecting the
hybridization complexes is carried out by detecting a fluorescent
label.
6. The method of claim 5, wherein the fluorescent label is
FITC.
7. The method of claim 1, wherein the hybridization complexes are
detected in interphase nuclei in the sample.
8. The method of claim 1, further comprising contacting the sample
with a reference probe which binds selectively to a sequence within
the centromere of the preselected chromosome.
9. A method of detecting an amplification at about position FLpter
0.85 on human chromosome 20, the method comprising:
contacting a chromosome sample from a patient with a composition
consisting essentially of one or more labeled nucleic acid probes
each of which binds selectively to a target polynucleotide sequence
at about position FLpter 0.85 on human chromosome 20 under
conditions in which the probe forms a stable hybridization complex
with the target sequence;
detecting the hybridization complex, thereby detecting the presence
or absence of the amplification.
10. The method of claim 8, wherein the step of detecting the
hybridization complex comprises determining the copy number of the
target sequence.
11. The method of claim 8, wherein the probe is labeled with
digoxigenin or biotin.
12. The method of claim 8, wherein the probe is selected from the
group consisting of polynucleotide sequences from cS20.10A1,
cS20.10B5, cS20.10H1, and cS20.10E2.
13. The method of claim 8, wherein the hybridization complex is
detected in interphase nuclei in the sample.
14. The method of claim 8, further comprising contacting the sample
with a reference probe which binds selectively to chromosome 20
centromere.
15. A composition comprising a nucleic acid probe which binds
selectively to a target polynucleotide sequence at about FLpter
0.85 on human chromosome 20.
16. The composition of claim 14, wherein the probe is labelled with
digoxigenin or biotin.
17. The composition of claim 14, wherein the probe is selected from
the group consisting of polynucleotide sequences from cS20.10A1,
cS20.10B5, cS20.10H1, and cS20.10E2.
Description
BACKGROUND OF THE INVENTION
Chromosome abnormalities are often associated with genetic
disorders, degenerative diseases, and cancer. In particular, the
deletion or multiplication of copies of whole chromosomes or
chromosomal segments, and higher level amplifications of specific
regions of the genome are common occurrences in cancer. See, for
example Smith, et al., Breast Cancer Res. Treat., 18: Suppl. 1:5-14
(1991, van de Vijer & Nusse, Biochim. Biophys. Acta. 1072:33-50
(1991), Sato, et al., Cancer. Res., 50: 7184-7189 (1990). In fact,
the amplification and deletion of DNA sequences containing
protooncogenes and tumor-suppressor genes, respectively, are
frequently characteristic of tumorigenesis. Dutrillaux, et al.,
Cancer Genet. Cytogenet., 49:203-217 (1990). Clearly the
identification of amplified and deleted regions and the cloning of
the genes involved is crucial both to the study of tumorigenesis
and to the development of cancer diagnostics.
The detection of amplified or deleted chromosomal regions has
traditionally been done by cytogenetics. Because of the complex
packing of DNA into the chromosomes, resolution of cytogenetic
techniques has been limited to regions larger than about 10 Mb;
approximately the width of a band in Giemsa-stained chromosomes. In
complex karyotypes with multiple translocations and other genetic
changes, traditional cytogenetic analysis is of little utility
because karyotype information is lacking or cannot be interpreted.
Teyssier, J. R., Cancer Genet. Cytogenet., 37:103 (1989).
Furthermore conventional cytogenetic banding analysis is time
consuming, labor intensive, and frequently difficult or
impossible.
More recently, cloned probes have been used to assess the amount of
a given DNA sequence in a chromosome by Southern blotting. This
method is effective even if the genome is heavily rearranged so as
to eliminate useful karyotype information. However, Southern
blotting only gives a rough estimate of the copy number of a DNA
sequence, and does not give any information about the localization
of that sequence within the chromosome.
Comparative genomic hybridization (CGH) is a more recent approach
to identify the presence and localization of amplified/deleted
sequences. See Kallioniemi, et al., Science, 258:818 (1992). CGH,
like Southern blotting, reveals amplifications and deletions
irrespective of genome rearrangement. Additionally, CGH provides a
more quantitative estimate of copy number than Souther blotting ,
and moreover also provides information of the localization of the
amplified or deleted sequence in the normal chromosome.
Generally, where detection of deletions or amplifications is
limited to the loss or gain of one copy of a sequence, the
resolution of prior art methods may be limited. New techniques
which provide increased sensitivity, more precise localization of
the affected DNA sequence, and more quantitative estimate of copy
number, even in samples of mixed normal and tumor cells is
particularly desirable. The present invention provides these and
other benefits.
SUMMARY OF THE INVENTION
The present invention provides methods and compositions for
detecting chromosome abnormalities (such as deletions and
amplifications) in a preselected chromosome. The methods comprise
providing a mapped library of labeled probes specific to the
chromosome; contacting a chromosome sample from a patient with the
library under conditions in which the probes bind selectively with
target polynucleotide sequences in the sample to form hybridization
complexes; detecting the hybridization complexes; and determining
the copy number of each complex.
If the selected chromosome is human chromosome 20 or 17, the
preferred libraries are as shown in Table 1 and Table 2,
respectively. The methods are typically carried out using
fluorescent in situ hybridization and the probes are labeled with
digoxigenin or biotin. The probes can be used to detect the target
sequences in interphase nuclei in the sample. A reference probe
which binds selectively to a sequence within the centromere of the
preselected chromosome can be used as a control.
Also provided are methods of detecting specific abnormalities
disclosed here. In particular, methods of detecting an
amplification at about position FLpter 0.85 on human chromosome 20
are disclosed. The methods comprise contacting a chromosome sample
from a patient with a composition consisting essentially of one or
more labeled nucleic acid probes each of which binds selectively to
a target polynucleotide sequence at about position FLpter 0.85 on
human chromosome 20 under conditions in which the probe forms a
stable hybridization complex with the target sequence; and
detecting the hybridization complex. The probes used preferably
comprise polynucleotide sequences from cS20.10A1, pk cS20.10B5,
cS20.10H1, or cS20.10E2.
Also provided are compositions comprising nucleic acid probes which
bind selectively to a target polynucleotide sequence at about
FLpter 0.85 on human chromosome 20. The probes may be labeled for
use in the methods of the invention.
The invention further provides kits for the detection of an
amplification at about position FLpter 0.85 on human chromosome 20.
The kits comprise a compartment which contains a nucleic acid probe
which binds selectively to a target polynucleotide sequence at
about FLpter 0.85 on human chromosome 20. The probes preferably
comprise polynucleotide sequences from cS20.10A1, cS20.10B5,
cS20.10H1, and cS20.10E2. The may further comprise Texas red avidin
and biotin-labeled goat anti-avidin antibodies.
DEFINITIONS
A "chromosome sample" as used herein refers to a tissue or cell
sample prepared for standard in situ hybridization methods
described below. The sample is prepared such that individual
chromosomes remain substantially intact and typically comprises
metaphase spreads or interphase nuclei prepared according to
standard techniques.
As used herein a "probe" is defined as a polynucleotide (either RNA
or DNA) capable of binding to a complementary target cellular
genetic sequence through one or more types of chemical bonds,
usually through hydrogen bond formation. It will be understood by
one of skill in the art that probes will typically substantially
bind target sequences lacking complete complementarity with the
probe sequence depending upon the stringency of the hybridization
conditions. The probes are preferably directly labelled as with
isotopes or indirectly labelled such as with biotin to which a
streptavidin complex may later bind. By assaying for the presence
or absence of the probe, one can detect the presence or absence of
the target. Probes of the invention will typically be between about
20 kb to about 60 kb, usually between about 30 and 50 kb.
A "composition consisting essentially of one or more probes each of
which binds selectively to a target polynucleotide sequence" refers
to a collection of one or more probes which bind substantially to
the target sequence and nowhere else in the target chromosome or
genome and which allow the detection of the presence or absence of
the target sequence. Such a composition may contain other nucleic
acids which do not materially affect the detection of the target
sequence. Such additional nucleic acids include reference probes
specific to a sequence in the centromere in the chromosome.
"Bind(s) substantially" refers to complementary hybridization
between an oligonucleotide and a target sequence and embraces minor
mismatches that can be accommodated by reducing the stringency of
the hybridization media to achieve the desired detection of the
target polynucleotide sequence.
"Hybridizing" refers the binding of two single stranded nucleic
acids via complementary base pairing.
"Nucleic acid" refers to a deoxyribonucleotide or ribonucleotide
polymer in either single- or double-stranded form, and unless
otherwise limited, would encompass known analogs of natural
nucleotides that can function in a similar manner as naturally
occurring nucleotides.
One of skill will recognize that the precise sequence of the
particular probes described herein can be modified to a certain
degree to produce probes that are "substantially identical" to the
disclosed probes, but retain the ability to bind substantially to
the target sequences. Such modifications are specifically covered
by reference to the individual probes herein. The term "substantial
identity" of polynucleotide sequences means that a polynucleotide
comprises a sequence that has at least 90% sequence identity, more
preferably at least 95%, compared to a reference sequence using the
methods described below using standard parameters.
Two nucleic acid sequences are said to be "identical" if the
sequence of nucleotides in the two sequences is the same when
aligned for maximum correspondence as described below. The term
"complementary to" is used herein to mean that the complementary
sequence is identical to all or a portion of a reference
polynucleotide sequence.
Sequence comparisons between two (or more) polynucleotides are
typically performed by comparing sequences of the two sequences
over a "comparison window" to identify and compare local regions of
sequence similarity. A "comparison window", as used herein, refers
to a segment of at least about 20 contiguous positions, usually
about 50 to about 200, more usually about 100 to about 150 in which
a sequence may be compared to a reference sequence of the same
number of contiguous positions after the two sequences are
optimally aligned.
Optimal alignment of sequences for comparison may be conducted by
the local homology algorithm of Smith and Waterman Adv. Appl. Math.
2:482 (1981), by the homology alignment algorithm of Needleman and
Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity
method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.)
85:2444 (1988), by computerized implementations of these
algorithms. These references are incorporated herein by
reference.
"Percentage of sequence identity" is determined by comparing two
optimally aligned sequences over a comparison window, wherein the
portion of the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) as compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which the
identical nucleic acid base or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
window of comparison and multiplying the result by 100 to yield the
percentage of sequence identity.
Another indication that nucleotide sequences are substantially
identical is if two molecules hybridize to the same sequence under
stringent conditions. Stringent conditions are sequence dependent
and will be different in different circumstances. Generally,
stringent conditions are selected to be about 5.degree. C. lower
than the thermal melting point (Tm) for the specific sequence at a
defined ionic strength and pH. The Tm is the temperature (under
defined ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. Typically, stringent
conditions will be those in which the salt concentration is at
least about 0.02 molar at pH 7 and the temperature is at least
about 60.degree. C.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the physical location of chromosome 20 specific
cosmids. The locations, determined by metaphase Fish and digital
image analysis, are shown as the mean of the Flpter (.+-.sem).
FIG. 2 shows the spot numbers of the different mapped, chromosome
20-specific cosmids (see FIG. 1) counted in interphase BT474 breast
cancer cells. A region around FLpter-0.85 is heavily amplified.
This region is therefore likely to contain a (proto)-oncogene.
FIG. 3 shows the physical locations on chromosome 17 of 40 cosmids
selected from the library LA17NC01. The locations, determined by
metaphase FISH and digital image analysis, are shown as the mean of
the FLpter (.+-.sem).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides methods and probe libraries useful
for detecting chromosome abnormalities in situ. In particular the
invention provides a means of identifying the presence of
multiplications or deletions in chromosomes and rapidly identifying
the chromosomal regions involved in those deletions or
multiplications.
Detection and Localization of Chromosomal Abnormalities
This invention is based on the use of libraries of genomic probes
in in situ hybridization to interphase nuclei or metaphase spreads
of chromosomes to detect and localize chromosomal abnormalities.
These abnormalities can be of several types, including extra or
missing individual chromosomes, extra or missing portions of a
chromosome (segmental duplications or deletions), breaks, rings and
chromosomal rearrangements. Chromosomal rearrangements include
translocations, dicentrics, inversions, insertions, amplification
and deletions.
Generally, the methods of the invention consist of two steps: 1)
The creation of a mapped library of probes, and 2) The in situ
hybridization of those probes to a chromosome and subsequent
detection of hybridization frequency to determine relative copy
number of a particular chromosomal region.
The mapped libraries of probes consist of a set of probes which
when hybridized to a normal chromosome are distributed relatively
uniformly across the region of interest. The region typically is
one chromosome or a part of one chromosome. In certain embodiments,
the library of probes can encompass the entire genome.
Each probe in the library is hybridized to the normal chromosome in
a metaphase spread in situ. The physical location of the probe on
the chromosome is determined by visualization of a marker, as
described in detail below. Probe locations are typically expressed
as the average fractional length from the p telomere (FLpter).
Once probes which hybridize to unique regions and show a relatively
uniform distribution have been identified and mapped, they may be
used to probe chromosomes of unknown genetic composition to
determine the presence or absence of amplifications or deletions
and other abnormalities. In particular, they may be used to probe
interphase nuclei which is the prevalent cell stage in most tissues
that are not actively dividing. Hybridization spots may be counted
by regular fluorescence microscopy to give the copy number as a
function of FLpter. The copy number relative to normal cells is
then indicative of various chromosome abnormalities such as
amplifications, deletions and the like.
Selection of a Chromosome
Typically, the probe libraries of the present invention are derived
from libraries spanning an entire chromosome. Alternatively,
libraries are constructed from multiple chromosomes, or from
regions spanning a segment of a chromosome. Single chromosomes may
be isolated by flow sorting using methods well known to those of
skill in the art. Briefly, chromosomes are isolated from cells
blocked in metaphase by the addition e.g., colcemid and stained
with two DNA-binding fluorescent dyes. The stained chromosomes are
then passed through a cell sorter and isolated using bivariate
analysis of the chromosomes by size and base pair composition (see,
e.g., Blennow et al., Hum. Genet. 90:371-374 (1992).
One of skill would recognize that the choice of a chromosome to map
may be influenced by prior knowledge of the association of a
particular chromosome with certain disease conditions. For example,
chromosome 17 is known to harbor several disease-linked genes
including p53, RARA, NF1, CMT and ERBB and there are reports
suggesting the presence of a tumor suppressor gene distal to p53
(e.g. Coles, et al. Lancet 336: 761-763.(1990), Cropp, et al. Proc.
Natl. Acad. Sci. USA 87:7737-7741.(1990) and Matsumura, et al.
Cancer Res. 52: 3474-3477 (1992)), a gene associated with early
onset breast cancer at 17q21 (Easton, et al. Am. J. Human Genet.,
52:678-701 (1993)) and amplification of one or more regions in
breast cancer. Kallioniemi, et al. Proc. Natl. Acad. Sci. USA
89:5321-5325 (1992).
Alternatively, whole genome screening techniques such as Southern
blotting, and Comparative Genome Hybridization (CGH) may be used to
identify chromosomes subject to frequent deletion and amplification
events and thus good candidates for further study using the present
invention. In particular CGH provides an effective means for
screening the genome for frequent deletion or amplification events.
CGH studies have indicated that sequences on chromosome 20q are
frequently amplified in both breast tumor cell lines and primary
breast tumors. Abnormalities can also be identified that are
suitable for prenatal screening.
In CGH, differently labeled test DNA and normal reference DNA are
hybridized simultaneously to normal chromosome metaphase spreads.
The hybridization is detected with two different fluorochromes.
Abnormal chromosomal regions containing duplications, deletions or
amplifications are detected as changes in the ratio of the two
fluorochromes along the target chromosomes. For a detailed
description of CGH see Kallioniemi, et al. Science, 258: 818-821
(1992).
One of skill would recognize that a library of the present
invention could be used to screen the entire genome. However
because of the high resolution of the technique and the large
number of probes required to screen the entire genome, CGH or other
methods are preferred for an initial screening.
Production of a Probe Library
In a preferred embodiment, a selected chromosome is isolated by
flow cytometry, as described above. The chromosome is then digested
with restriction enzymes appropriate to give DNA sequences of at
least about 20 kb and more preferably about 40 kb. Techniques of
partial sequence digestion are well known in the art. See, for
example Perbal, A Practical Guide to Molecular Cloning 2nd Ed.,
Wiley New York (1988) incorporated herein by reference. The
resulting sequences are ligated with a vector which contains a
resistance marker. The vector is transfected into and propagated in
the appropriate host. Exemplary vectors suitable for this purpose
include cosmids, yeast artificial chromosomes (YACs), bacterial
artificial chromosomes (BACs) and P1 phage. Typically, cosmid
libraries are prepared. The cosmid library then consists of single
clones of the transfected bacteria.
While it is possible to generate cosmid libraries, as described
above, libraries spanning entire chromosomes are available
commercially (Clonetech, South San Francisco, Calif.) or from the
Los Alamos National Laboratory. For example, the Los Alamos
supplies a library designated LA17NC01 which comprises a set of
inserts in cosmids that span the entire chromosome 17 sorted from
the mouse-human hybrid cell line, 38L-27. The Los Alamos library
for chromosome 20 is designated LA20NC01.
The cosmid probes must be labeled for use in in situ hybridization.
The probes may be detectably labeled prior to the hybridization
reaction. Alternatively, a detectable label may be selected which
binds to the hybridization product. Probes may be labeled with any
detectable group for use in practicing the invention. Such
detectable group can be any material having a detectable physical
or chemical property. Such detectable labels have been
well-developed in the field of immunoassays and in general most any
label useful in such methods can be applied to the present
invention. Thus a label is any composition detectable by
spectroscopic, photochemical, biochemical, immunochemical, or
chemical means. Useful labels in the present invention include
fluorescent dyes, electron-dense reagents, enzymes (as commonly
used in an ELISA), biotin, dioxigenin, or haptens and proteins for
which antisera or monoclonal antibodies are available. The
particular label used is note critical to the present invention, so
long as it does not interfere with the in situ hybridization of the
probe. In addition the label must be detectible in as low copy
number as possible thereby maximizing the sensitivity of the assay
and yet be detectible above any background signal. Finally, a label
must be chosen that provides a highly localized signal thereby
providing a high degree of spatial resolution when physically
mapping the probe against the chromosome. In a preferred
embodiment, the label is digoxigenin-11-dUTP or biotin-14-dATP,
which are then detected using fluorophores.
The labels may be coupled to the probes in a variety of means known
to those of skill in the art. In a preferred embodiment the probe
will be labeled using nick translation or random primer extension
(Rigby, et al. J. Mol. Biol., 113:237 (1977) or Sambrook, et al.,
Molecular Cloning--A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1985)).
Mapping of Probe Library
Once a probe library is constructed, a subset of the probes is
physically mapped on the selected chromosome. FISH and digital
image analysis can be used to localize cosmids along the desired
chromosome. This method is described in detail below and in Lichter
et al., Science, 247:64-69 (1990). Briefly, the clones are mapped
by FISH to metaphase spreads from normal cells using e.g., FITC as
the fluorophore. The chromosomes are counterstained by a stain
which stains DNA irrespective of base composition (e.g., propidium
iodide), to define the outlining of the chromosome. The stained
metaphases are imaged in a fluorescence microscope with a
polychromatic beam-splitter to avoid color-dependent image shifts.
The different color images are acquired with a CCD camera and the
digitized images are stored in a computer. A computer program is
then used to calculate the chromosome axis, project the two (for
single copy sequences) FITC signals perpendicularly onto this axis,
and calculate the average fractional length from a defined
position, typically the p-telomere.
The accuracy of the mapped positions of the probes can be increased
using interphase mapping. Briefly, the distance between two probes
which are found by metaphase mapping to be very close in measured
in normal interphase nuclei. The genomic distance between the two
is equal to the square of the physical distance (Van den Engh et
al., Science 257:1410 (1992)). If the order is uncertain, the
probes are labeled with different colors and their relative
distance to a third (distant) probe. Trask et al., Am. J. Hum.
Genet. 48:1 (1991).
Typically, a mapped library will consist of between about 20 and
about 125 clones, more usually between about 30 and about 50
clones. Ideally, the clones are distributed relatively uniformly
across the region of interest, usually a whole chromosome.
In situ Hybridization with Mapped Library
The mapped library is then used to screen for chromosomal
abnormalities in a sample. In the methods of the invention, a
chromosome sample (typically either a metaphase spread or
interphase nuclei) is analyzed using standard in situ hybridization
techniques. Several guides to the techniques are available, e.g.,
Gall et al. Meth. Enzymol., 21:470-480 (1981) and Angerer et al. in
Genetic Engineering: Principles and Methods Setlow and Hollaender,
Eds. Vol 7, pgs 43-65 (plenum Press, New York 1985).
Briefly, a chromosomal sample is prepared by depositing cells,
either as single cell suspensions or as tissue preparation, on
solid supports such as glass slides and fixed by choosing a
fixative which provides the best spatial resolution of the cells
and the optimal hybridization efficiency.
Generally, in situ hybridization comprises the following major
steps: (1) fixation of tissue or biological structure to analyzed;
(2) prehybridization treatment of the biological structure to
increase accessibility of target DNA, and to reduce nonspecific
binding; (3) hybridization of the mixture of nucleic acids to the
nucleic acid in the biological structure or tissue; (4)
posthybridization washes to remove nucleic acid fragments not bound
in the hybridization and (5) detection of the hybridized nucleic
acid fragments. The reagent used in each of these steps and their
conditions for use vary depending on the particular
application.
In some applications it is necessary to block the hybridization
capacity of repetitive sequences. In this case, human genomic DNA
is used as an agent to block such hybridization. The preferred size
range is from about 200 bp to about 1000 bases, more preferably
between about 400 to about 800 bp for double stranded, nick
translated nucleic acids.
Hybridization protocols for the particular applications disclosed
here are described in detail below. Suitable protocols are
described in Pinkel et al. Proc. Natl. Acad. Sci. USA, 85:9138-9142
(1988) and in EPO Pub. No. 430,402.
Standard in situ hybridization techniques are used to probe a given
sample. Hybridization protocols for the particular applications
disclosed here are described in detail below. Suitable protocols
are described in Pinkel et al. Proc. Natl. Acad. Sci. USA,
85:9138-9142 (1988) and in EPO Pub. No. 430,402.
Typically, it is desirable to use dual color FISH, in which two
probes are utilized, each labelled by a different fluorescent dye.
A test probe that hybridizes to the region of interest is labelled
with one dye, and a control probe that hybridizes to a different
region is labelled with a second dye. A nucleic acid that
hybridizes to a stable portion of the chromosome of interest, such
as the centromere region, is often most useful as the control
probe. In this way, differences between efficiency of hybridization
from sample to sample can be accounted for.
The FISH methods for detecting chromosomal abnormalities can be
performed on nanogram quantities of the subject nucleic acids.
Paraffin embedded tumor sections can be used, as can fresh or
frozen material. Because FISH can be applied to the limited
material, touch preparations prepared from uncultured primary
tumors can also be used (see, e.g., Kallioniemi, A. et al.,
Cytogenet. Cell Genet. 60: 190-193 (1992)). For instance, small
biopsy tissue samples from tumors can be used for touch
preparations (see, e.g., Kallioniemi, A. et al., Cytogenet. Cell
Genet. 60: 190-193 (1992)). Small numbers of cells obtained from
aspiration biopsy or cells in bodily fluids (e.g., blood, urine,
sputum and the like) can also be analyzed. For prenatal diagnosis,
appropriate samples will include amniotic fluid and the like.
Once a region of interest has been identified and mapped with the
methods of the invention, one of skill will recognize that there
are numerous means of identifying and/or screening for this region.
The region may be sequenced by digesting chromosomal DNA with
restriction enzymes and identifying the specific
duplication-bearing fragments using the mapped cosmids of the
invention as hybridization probes. The positive clones may then be
subcloned into appropriate vectors and sequenced.
Sequence information permits the design of highly specific
hybridization probes or amplification primers suitable for
detection of the target sequences. This is useful for diagnostic
screening systems as well as research purposes.
Means for detecting specific DNA sequences are well known to those
of skill in the art. For instance, oligonucleotide probes chosen to
be complementary to a select subsequence with the region can be
used. Alternatively, sequences or subsequences may be amplified by
a variety of DNA amplification techniques (for example via
polymerase chain reaction, ligase chain reaction, transcription
amplification, etc.) prior to detection using a probe.
Amplification of DNA increases sensitivity of the assay by
providing more copies of possible target subsequences. In addition,
by using labeled primers in the amplification process, the DNA
sequences may be labeled as they are amplified.
The following example is provided to illustrate but not limit the
present invention.
EXAMPLE 1
Mapping of Chromosome 20
Results of experiments employing "Comparative Genome Hybridization"
(CGH) to screen the whole genome for amplifications, indicate that
sequences on chromosome 20q are frequently amplified in both breast
tumor cell lines and primary breast tumors. In order to define
these genetic alterations in more detail, a library of cosmid FISH
probes was isolated and physically mapped to chromosome 20. The
library of mapped probes could then be used to probe chromosome 20
using FISH to determine the particular loci involved in
amplifications and deletions. This example details the creation of
the library of probes physically mapped to chromosome 20.
Cosmids from a chromosome 20 library were isolated at random from
single bacterial clones using Qiagen columns according to the
manufacturers instructions (Qiagen Inc., Chatsworth, Calif.).
Cosmid DNA was labeled by nick-translation with biotin-14-dATP
(Gibco), to give fragments of length 0.3-1.0 kb (under
non-denaturing conditions). These probes were hybridized to normal
human lymphocyte metaphase preparations. The slides were denatured
at 70.degree. C. for 3 minutes in 70% formamide/2X SSC, followed by
dehydration in 70%/85%/100% ethanol. The slides were hybridized
overnight with 40 ng of human biotin-labeled cosmid DNA in the
presence of 5 .mu.g human placental DNA (Sigma) in 10 .mu.l 50%
formamide/2X SSC at 37.degree. C. The probe was denatured at
70.degree. C. for 5 minutes and allowed to renature at 37.degree.
C. prior to application to the slides. The slides were washed three
times in 50% formamide/2X SSC, once in 0.1X SSC, and twice in 2X
SSC at 45.degree. C. (15 minutes for each wash). The remaining
steps were performed at room temperature.
The slides were equilibrated in 4X SSC/0.1% Triton X100 ("wash"
buffer), and blocked for 5 minutes in wash buffer with 5% dry
milk/0.1% Sodium Azide ("block" buffer). Staining for biotinylated
probe was done with 5 .mu.g/ml avidin-FITC (60 minutes, in block
buffer), amplified by 30 minutes incubation with 5 .mu.g/ml
biotinylated anti-avidin (in block buffer), and another 30 minutes
incubation with avidin-FITC. The slides were then washed three
times for 10 minutes each after each staining step. The slides were
equilibrated in 0.1X SSC prior to application of anti-fade solution
(ref) with 0.05 .mu.g/ml propidium iodide and 0.4 .mu.M DAPI.
The slides were first inspected in a fluorescence microscope to
determine whether signals were present (29/40 cosmids tested) and,
if so, whether the cosmid detected single-copy sequences (28/40
cosmids tested). One cosmid hybridized to the centromere of
chromosome 20, but also hybridized to the p-arms of acrocentric
chromosomes. Metaphases hybridized with cosmids detecting single
copy sequences were analyzed in a Nikon SA fluorescence microscope
equipped with a CCD camera (Photometrics Inc., Tucson, Ariz.) and a
polychromatic beamsplitter (Chroma Technology Inc.,
Brattleborrough, Vt.) to avoid color-dependent image shifts. The
images of chromosome 20 were analyzed with computer software to
determine the mean position (of the two FITC probe spots) in term
of fractional length from the p telomere (FLpter) along the
chromosome axis as defined by propidium iodide staining using the
methods generally described by Lichter et al., supra.
Several metaphases were analyzed for each cosmid, the FLpter value
and the SEM are given in Table 1. The average SD of all the FLpter
values was 0.030, corresponding to -2 Mb. Cosmids which are
separated by a FLpter value of less than 2.5 times the SEM could
not be ordered with statistical confidence. The vertical lines in
Table 1 span cosmids which could not be ordered reliably.
TABLE 1 ______________________________________ FLpter values of
chromosome 20 cosmids. Cosmid FLpter n SEM
______________________________________ ##STR1## ##STR2## ##STR3##
______________________________________ *Any two cosmids connected
by a vertical line cannot be ordered with statistical
confidence.
The FLpter values are graphically illustrated in FIG. 1, together
with an ideogram of chromosome 20. A chromosome 20 centromere probe
(p3-4) mapped to FLpter=0.443 (SEM=0.008, n=10), in good agreement
with the position of the centromere in this ideogram and the
reported physical location of the chromosome 20 centromere
(Schnittger, et al. Genomics, 16:50-55 (1993); Passarge, E. pp.
135-205 In Methods in Human Genetics Schwarzacher & Wolf, Eds.
Springer, Berlin (1974). The corresponding band locations indicated
in FIG. 1 were also confirmed visually relative to the position of
DAPI bright bands (corresponding approximately to Giemsa-stained
bands under these conditions). The cosmids seem to be fairly evenly
distributed over the whole chromosome, with possible under- and
over-representation in the centromere region and the region with a
FLpter value of -0.6, respectively.
Mapping Chromosome 17
The chromosome 17 cosmid library (designated LA17NC01) was prepared
at the Los Alamos National Laboratory from chromosomes flow sorted
from the mouse-human hybrid cell line, 38L-27. The sorted
chromosomes were examined for purity using FISH. DNA was extracted,
partially digested with Sau3A1, dephosphorylated, and cloned into
sCosl with HB101 as the host. Characterization of the initial
library showed it to have 39X representation with 92% human
inserts, 2.6% mouse and 5.4% non-recombinant.
Approximately 12,000 colonies were picked and grown in microtiter
plates to provide a 5X coverage of chromosome 17. The contents of
one set of these plates were pooled together to provide a pooled 5X
library. For this study, the 5X library was plated and 288
individual clones were picked at random and arrayed in three
microtiter plates. Seventy one of these were analyzed using FISH.
Twenty cosmid probes previously mapped by genetic linkage analysis
(O'Connell, et al. Genomics, 15:38-47 (1993) also were selected for
analysis.
FISH was performed essentially as described previously. See
Kallioniemi, et al. Proc. Natl. Acad. Sci. USA, 89:5321-5325 (1992)
and Pinkel, et al., Proc. Natl. Acad. Sci. USA, 85:9138-9142 (1988)
both of which are incorporated herein by reference. Briefly, DNA
was isolated from individual clones and from the 5X pool using
Qiagen columns (Qiagen Inc. Chatsworth, Calif.) according to the
manufacturers instructions. DNA was labeled using nick translation
with biotin-14-dATP. DNA from a chromosome 17 centromeric repeat
probe was labeled with digoxigenin-11-dUTP. Each probe was
hybridized along with the chromosome 17 centromere probe to
metaphase spreads prepared from normal peripheral blood
lymphocytes. Hybridized probes were detected using Avidin-Texas Red
and anti-digoxigenin-FITC. Metaphase chromosomes were
counterstained using DAPI in an anti-fade solution.
Analysis of the samples was accomplished using a digital image
analysis system as described above. A semi-automated program was
used to 1) segment each DAPI image, 2) define the chromosome medial
axis, 3) define the center of mass of each candidate hybridization
domain, and 4) calculate the fractional location of each domain
along the chromosome axis relative to the telomere of the short arm
of the chromosome (FLpter). Candidate hybridization domains defined
by the analysis program were confirmed by visual inspection. A
chromosome 17 centromeric probe was used in each hybridization as
an internal reference. If the FLpter value of the centromere did
not fall within the expected range (0.300<FLpter<0.342), the
chromosome was not used for FLpter calculation.
FISH using DNA from the whole library (LA17NC01) as a painting
probe resulted in intense, specific staining of the entire
chromosome 17 indicating that all portions of the chromosome are
represented in the library. Seventy-one individual cosmids selected
from the library were roughly mapped using FISH. Of these, 15 (21%)
mapped to the p-arm, 46 (65%) to the q-arm, 2 (3%) to the
peri-centromeric repeat region. This distribution is approximately
that expected for randomly distributed probes. Eight cosmids (11%)
gave no signal on any human chromosome and so may have been of
non-human origin. This is consistent with the initial library
characterization by slot blot analysis that showed 8% of the clones
to be non recombinant or to contain a mouse insert.
In order to identify probes that would be useful for FISH analysis
of interphase cells, the 40 cosmids that gave the most distinctive
hybridization signals both in metaphase and interphase were mapped
using digital image analysis. FLpter values are listed in Table 2
for each cosmid. The standard deviation (sd) for the FLpter
measurements and the number of chromosomes analyzed for each probe
(n) are listed to permit calculation of the standard error of the
mean (sem=sd.sqroot./n) for each FLpter estimate. Probes whose
FLpter means differ by >2.5 sem can be ordered with statistical
confidence.
FIG. 3 shows that the probes are distributed over the whole
chromosome, although there seems to be a slight over-representation
of sequences near 17p11-p12. Two color, pair-wise hybridizations to
metaphase chromosomes showed that these did not represent the same
clone. FLpter values are related to the ICSN chromosome 17 ideogram
in FIG. 3 since other studies have shown a reasonable
correspondence between FLpter value and band location (Lawrence, et
al., Science, 249:928-932 (1990); Lichter, P., et al. Science
247:64-69. (1990)). However, this relation should be considered
only approximate since band locations on the ideograms are
inexact.
Twenty cosmids that had also been mapped by genetic linkage
analysis (O'Connell, et al. Genomics, 15:38-47 (1993)) were also
physically mapped. The average standard deviation of the FLpter
measurements for all cosmids was 0.035. This corresponds to about 3
Mbs. The average mapping precision determined as the standard error
of the mean (sem) was about 0.01 corresponding to .about.1 Mb.
Cosmids separated by .about.2.5 Mb (i.e. whose means are separated
by >2.5 sem in Table 2) can be ordered with statistical
confidence.
Detection of Amplifications in Chromosome 20
FIG. 2, shows an example where the spot numbers of the different
mapped, chromosome 20-specific cosmids (see FIG. 1) were counted in
interphase BT474 breast cancer cells using standard FISH techniques
as described above. A region around FLpter-0.85 is heavily
amplified.
All of the references cited herein are hereby incorporated by
reference. For the purposes of clarity and understanding, the
invention has been described in these examples and the above
disclosure in some detail. It will be apparent, however, that
certain changes and modifications may be practiced within the scope
of the appended claims.
TABLE 2
__________________________________________________________________________
FLpter values measured for 60 cosmids including 20 that were mapped
previously by genetic linkage analysis (shown in bold type). Gen
Gen Probe FLpter sd n loc (cM) Probe FLpter sd n loc (cM)
__________________________________________________________________________
cK17.79 0.025 0.018 5 cK17.16 0.564 0.048 8 cLS17.6 0.066 0.037 25
13 fLB17.20 0.580 0.046 21 90 fLB17.8 0.068 0.037 16 15 cK17.11
0.591 0.035 12 cK17.29 0.068 0.031 17 cLS17.13 0.594 0.040 22 90
fLB17.9 0.073 0.037 20 19 cK17.87 0.598 0.026 8 fLB17.16 0.076
0.041 28 15-18* cK17.30 0.618 0.041 9 cK17.22 0.122 0.020 11
cK17.76 0.657 0.019 12 cK17.88 0.177 0.042 9 cK17.84 0.666 0.024 9
cK17.31 0.197 0.036 10 cK17.73 0.714 0.037 11 cK17.24 0.199 0.028
11 cK17.33 0.728 0.039 13 cK17.80 0.205 0.035 11 fLB17.4 0.773
0.033 20 111-123* cK17.17 0.219 0.044 8 cK17.72 0.814 0.037 11
cK17.83 0.220 0.043 10 cK17.14 0.823 0.026 11 cK17.19 0.224 0.031
10 fLB17.14 0.825 0.033 25 120 pYNM67 0.233 0.057 15 59-63* cK17.28
0.837 0.052 16 cK17.81 0.236 0.037 11 cK17.53 0.867 0.039 15
fLB17.5 0.251 0.040 17 61-64 cK17.12 0.871 0.023 12 cK17.23 0.306
0.029 4 cK17.54 0.894 0.032 16 cK17.75 0.355 0.038 9 cK17.15 0.919
0.035 12 fLB17.18 0.410 0.035 20 77 c1-26 0.922 0.035 18 144
cK17.37 0.411 0.033 5 fLB17.17 0.942 0.037 25 148-151* cK17.18
0.433 0.044 12 cK17.27 0.943 0.032 13 cK17.32 0.446 0.030 9 cEFD52
0.950 0.045 15 167 fLB17.6 0.451 0.040 18 77-80* cK17.71 0.953
0.028 11 cK17.89 0.470 0.048 12 cLS17.9 0.962 0.027 21 159 cK17.86
0.510 0.035 8 fLB17.7 0.964 0.026 24 165-169* cK17.25 0.515 0.023 8
fLB17.2 0.969 0.030 28 166-170* cK17.38 0.537 0.031 14 fLB17.10
0.985 0.016 23 177 cK17.74 0.539 0.036 9 cK17.77 0.989 0.015 9
fLB17.1 0.557 0.038 19 88 cK17.78 0.994 0.005 12
__________________________________________________________________________
*Genetic locations for these probes were estimated graphically from
information presented by 'Connell, et al. Genomics, 15: 38-47
(1993).
* * * * *